US9796996B2 - Means and methods for distinguishing FECV and FIPV - Google Patents

Means and methods for distinguishing FECV and FIPV Download PDF

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US9796996B2
US9796996B2 US13/522,863 US201113522863A US9796996B2 US 9796996 B2 US9796996 B2 US 9796996B2 US 201113522863 A US201113522863 A US 201113522863A US 9796996 B2 US9796996 B2 US 9796996B2
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Petrus Josephus Marie Rottier
Hui-Wen Chang
Herman F. Egberink
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Universiteit Utrecht Holding BV
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the field of veterinary diagnosis, more specifically the invention relates to the field of feline coronaviruses and identification thereof.
  • FCoVs Feline coronaviruses
  • FCoVs Feline coronaviruses
  • FCoV are common pathogens of domestic and non-domestic Felidae, including but not limited to cats, lions, tigers, leopards, jaguars, lynxes, caracals, cheetahs, cougars and servals.
  • FCoV are closely related to canine coronavirus (CCoV) and transmissible gastroenteritis virus (TGEV) of swine.
  • TCoV canine coronavirus
  • TGEV transmissible gastroenteritis virus
  • Two serotypes, I and II exist of FCoV of which serotype I predominates, with 80-95% of FCoV infections.
  • Type II FCoV presumably results from RNA recombination in animals doubly infected by serotype I FCoV and CCoV, during which a CCoV spike gene or part thereof is incorporated into the FCoV genome, apparently an infrequently occurring event.
  • Feline enteric coronavirus FECV
  • FECV Feline enteric coronavirus
  • FECV is the most common pathotype of FCoV, for both serotype I and serotype II.
  • FECV is mainly confined to the intestines, spreads via the oral-fecal route, and is highly contagious. FECV infection generally occurs unapparently; sometimes, however, it causes symptoms such as mild enteritis (Haijema et al., 2007).
  • feline infectious peritonitis a (then) rare but serious disease in cats, was reported to be caused by a feline coronavirus, which was called feline infectious peritonitis virus (FIPV). Contrary to FECV, FIPV is highly virulent. FIPV infection can be either granulomatous (dry) or effusive (wet) and is a progressive and usually fatal disease. Symptoms of FIP include failure to thrive in young cats, lameness, fluctuating fever, inappetence and weight loss resulting in death (Pedersen 2009).
  • FIPV originates from FECV by de novo mutation in infected felines resulting in a highly virulent FIP virus.
  • the mutation giving rise to FIPV has not been identified but has been proposed to be in the non structural 3c, 7a or 7b genes (see FIG. 1 ), which encode proteins with unknown function (Vennema et al., 1998, Tru et al., 1996, Kennedy et al., 2001, Pedersen 2009).
  • FECV is confined to the intestines
  • FIPV which is able to infect macrophages and monocytes
  • Dye and Siddell found 100% nucleotide identity and thus questioned the mutation hypothesis according to which the liver coronavirus is a mutated jejunum coronavirus. They suggested that in cats suffering from FIP the same virulent feline coronavirus strain was present in both liver and jejunum.
  • the present inventors identified a number of differences in the spike protein of tissue culture-adapted serotype II feline coronaviruses FECV 79-1683 and FIPV 79-1146 (Rottier et al., 2005).
  • the FIPV 79-1146 contained several mutations in the C-terminal domain of the spike protein, the S2 domain.
  • FECV 79-1683 and FIPV 79-1146 are not prototypical feline coronaviruses and are thus not representative for the serotype I FECV and FIPV most cats are infected with (Pedersen 2009).
  • the serotype II feline coronaviruses originate from RNA recombination of canine and feline coronaviruses and contain the canine coronavirus spike protein. Spike proteins of feline and canine coronaviruses have only approximately 45% amino acid sequence identity (Motokawa et al., 1996).
  • FECV 79-1683 and FIPV 79-1146 are tissue culture-adapted to cell lines other than macrophages. Because FIPV infects monocytes and macrophages in vivo, tropism of these laboratory strains differs from prototypical feline coronaviruses.
  • FIPV 79-1146 unlike serotype I FIPV which infects monocytes and macrophages, is exceptionally virulent by every common route of infection (Pedersen 2009).
  • FECV 79-1683 cannot be qualified as a true FECV as argued extensively by Pedersen in his recent review (Pedersen, 2009).
  • FECV 79-1683 lacks most of the 7b gene, which is present in non-tissue culture-adapted strains of FECV and has a deleterious mutation in its 3c gene, indicating that it may have originated from an FIPV.
  • Feline coronavirus infection is generally demonstrated by the presence of antibodies in the blood.
  • An effective treatment or vaccine for FIPV infection does not exist.
  • a commercially available vaccine consisting of a temperature sensitive mutant of a FIPV strain has not convincingly proven its protective efficacy in a number of immunization studies (McArdle et al., 1995; Fehr et al., 1997).
  • a further complicating factor is that the clinical picture of FIP is highly variable and, as a consequence, the disease cannot easily be established unequivocally.
  • the diagnosis is often a presumptive one, based on anamnestic, clinical and non-specific laboratory parameters. Because there is no specific diagnostic test for FIPV, it is often also not possible to discriminate between FIP and other diseases with overlapping symptoms. Both diagnostic tests for and vaccines against FIPV are highly needed due to the progressive and debilitating course of FIP.
  • FIPV harbours a specific alteration relative to FECV in the spike protein at amino acid position 1049 as depicted in FIG. 2B (SEQ ID NO:8 and SEQ ID NO:9).
  • the invention therefore provides a method for identifying feline infectious peritonitis virus (FIPV) comprising determining the identity of an amino acid of a feline coronavirus spike protein at a position corresponding to amino acid position 1049 as depicted in FIG. 2B (SEQ ID NO:8 and SEQ ID NO:9), and identifying the feline coronavirus as FIPV if the determined identity of the amino acid is not a methionine.
  • FECV is identified if the determined identity of the amino acid is methionine.
  • identifying FIPV or FECV is meant the identification of a virulent (FIPV) or an avirulent (FECV) type feline coronavirus. Identification is carried out by determining the identity of an amino acid and/or nucleic acid sequence of said feline coronavirus.
  • a feline coronavirus nucleic acid sequence comprises a chain of nucleotides, preferably (c)DNA or RNA, that is part of a feline coronavirus or obtained from a feline coronavirus, either directly, or after processing, such as for example by using reverse transcriptase PCR, and/or amplification.
  • a feline coronavirus spike protein is a feline coronavirus membrane protein comprising an ectodomain.
  • the spike protein is one of the four canonical structural proteins of coronavirus and is responsible for attachment to and entry of the virus into cells during infection.
  • FIPV from lesions of cats with pathologically confirmed FIP were compared genetically with FECV obtained from asymptomatic cats.
  • Typical lesions of FIP were (pyo)granulomatous lesions presented in different internal organs mainly in spleen, liver, lung, kidney, or mesenteric lymph node. Due to the high mutation rates of RNA viruses, numerous differences were observed between individual FECV and FIPV sequences.
  • the amino acid at position 1049 of the spike protein as depicted in FIG. 2B is a methionine
  • FIPV lesion isolates an alteration of the amino acid at position 1049 as depicted in FIG.
  • the nucleic acid sequence encoding the methionine at position 1049 of the spike protein of FECV corresponds to the codon comprising nucleotide positions 3145, 3146 and/or 3147 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • the nucleotide sequence encoding the methionine in the FECV spike protein at position 1049 as depicted in FIG. 2A is adenine-thymine-guanine (a-t-g), which corresponds with the sequence adenine-uridine-guanine (a-u-g) in the viral genomic RNA.
  • any substitution of at least one nucleotide in this nucleotide codon results in an amino acid other than methionine in the spike protein of FECV at position 1049 as depicted in FIG. 2B .
  • the identified nucleotide and/or amino acid sequence at nucleotide positions 3145, 3146 and/or 3147 of the gene encoding a feline coronavirus spike protein, and amino acid position 1049 respectively as depicted in FIGS. 2A and 2B is used to discriminate between FIPV and FECV.
  • a polymorphism of the feline coronavirus that enables distinguishing FECV and FIPV has been identified in prototypical serotype I FECV and FIPV.
  • the present inventors further found that a significant part of the small percentage of FIPV which do not harbour the specific alteration relative to FECV in the spike protein at amino acid position 1049 as depicted in FIG. 2B , harbours a specific alteration relative to FECV in the spike protein at amino acid position 1051 as depicted in FIG. 2B . In these cases, a serine at this position appeared to be substituted. Thus, the specific alteration at amino acid position 1051 also provides an approach to identify FIPV.
  • the invention therefore also provides a method for identifying feline infectious peritonitis virus (FIPV) comprising determining the identity of an amino acid of a feline coronavirus spike protein at a position corresponding to amino acid position 1051 as depicted in FIG. 2B , and identifying the feline coronavirus as FIPV if the determined identity of the amino acid is not a serine.
  • FIPV feline infectious peritonitis virus
  • Also provided is a method for determining whether feline infectious peritonitis virus (FIPV) is present in a sample comprising determining whether said sample comprises a feline coronavirus, and if a feline coronavirus is present determining the identity of an amino acid in a spike protein of said feline coronavirus at a position corresponding to amino acid position 1051 as depicted in FIG. 2B , and determining that FIPV is present if said amino acid is not serine.
  • FIPV feline infectious peritonitis virus
  • the nucleic acid sequence encoding the serine at position 1051 of the spike protein of FECV corresponds to the codon comprising nucleotide positions 3151, 3152 and 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • Serine is encoded by the nucleotide codons t/u-c-t/u, t/u-c-c, t/u-c-a, t/u-c-g, c-g-t/u and c-g-c.
  • any substitution of one or more nucleotides in this nucleotide codon resulting in a nucleotide sequence other than these codons results in an amino acid other than serine in the spike protein of FECV at position 1051 as depicted in FIG. 2B .
  • the identified nucleotide and/or amino acid sequence at either nucleotide positions 3151, 3152 and/or 3153 of the gene encoding a feline coronavirus spike protein, and/or amino acid position 1051 of the Spike protein respectively, as depicted in FIGS. 2A and 2B is also used to discriminate between FIPV and FECV.
  • an alteration of a serine at an amino acid position corresponding to position 1051 as depicted in FIG. 2B is the result of a replacement of the nucleobase thymine at a position corresponding to nucleotide position 3151 as depicted in FIG. 2A with the nucleobase guanine.
  • the identity of the amino acids in a spike protein of a feline coronavirus at positions corresponding to amino acid positions 1049 and 1051 as depicted in FIG. 2B are both determined. If the identity of both these amino acids is determined, a high accuracy in distinguishing FIPV and FECV is obtained. In one embodiment, the identity of the amino acids at positions 1049 and 1051 as depicted in FIG. 2B is determined in one test. However, it is also possible to determine the identity of these amino acid sequentially. For instance, first the identity of the amino acid at a position corresponding to position 1049 as depicted in FIG. 2B is determined.
  • the identity of the amino acid at a position corresponding to position 1051 as depicted in FIG. 2B is preferably determined. If the presence of an amino acid other than a methionine is detected at this position, determining the identity of the amino acid at a position corresponding to position 1051 as depicted in FIG. 2B can be omitted. However, the identity of the amino acid at this position may also be determined in that case.
  • a nucleic acid sequence of the spike gene (nucleotides 1-4407) of feline coronavirus comprising the nucleotides 20395-24801 as defined in the sequence of gene accession number NC_012955 (Feline coronavirus UM10, complete genome) and nucleotides 20382-24788 as defined in the sequence of gene accession number NC_012952 (Feline coronavirus UU8, complete genome) is presented in FIG. 2A .
  • Nucleotides 20395-24801 of NC_012955 encode the feline coronavirus spike protein (YP_003038574).
  • Nucleotides 20382-24788 of NC_012952 encode the feline coronavirus spike protein (YP_003038543).
  • nucleotides 3145, 3146 and 3147 of the gene encoding the spike protein as used throughout the description and as depicted in FIG. 2A correspond to nucleotides 23539, 23540 and 23541 of the complete genome as defined in the sequence of NC_012955 and/or nucleotides 23526, 23527 and 23528 of the complete genome as defined in the sequence of NC_012952.
  • the amino acid sequence of a feline coronavirus spike protein referring to the amino acid numbering defined in the sequences of YP_003038574 and YP_003038543 which are partial translations of NC_012955 and NC_012952 respectively is presented in FIG. 2B .
  • the numbering of amino acid positions as used throughout the description refers to the amino acid positions as defined in YP_003038574 and/or YP_003038543.
  • a skilled person is able to identify the nucleotide and amino acid positions in any given feline coronavirus sequence which correspond to the nucleotide positions 3145, 3146 and/or 3147 and amino acid position 1049 and the nucleotide positions 3151, 3152 and/or 3153 and amino acid position 1051 as depicted in FIG. 2A or 2B , for instance using alignment software such as “Align 2” or “Bioconductor”.
  • the symptoms of FIP include for instance the accumulation of ascitic fluid within the abdomen (only in effusive FIP), retarded growth, lack of appetite, fever, weight loss and diarrhea. As indicated herein above, similar symptoms are also observed with cats suffering from other diseases, making unequivocal diagnosis of FIP so far impossible. Now that a polymorphism has been identified for feline coronavirus spike protein that allows for determining the presence of FIPV in a sample it can be determined whether a feline, for instance a cat, suffers from FIP. Because currently there is no treatment for FIP, and the course of the disease is progressive and debilitating resulting inevitably in death, it can be decided to euthanize said cat when the animal has been demonstrated to carry FIPV.
  • the cat or cattery owner can take proper measures to prevent possible spread of the infection and/or reduce predisposing conditions such as stress.
  • FIPV has been demonstrated to be absent in a cat
  • feline infectious peritonitis can be eliminated as a possible cause of the disease. Therefore, in that case the cat should not be euthanized but diagnostic approaches could be continued and the animal could be provided with treatment for the possible alternative disease(s) the symptoms of which resemble those of FIP.
  • Such treatment can for instance be further symptomatic treatment or application of antibiotics to counteract a possible bacterial cause of the disease.
  • feline infectious peritonitis virus FIPV
  • a method for determining whether feline infectious peritonitis virus comprising preferably from a feline or from a substance that has been in contact with a feline, determining whether said sample comprises a feline coronavirus and, if a feline coronavirus is present, determining the identity of an amino acid in a spike protein of said feline coronavirus at a position corresponding to amino acid position 1049 and/or 1051 as depicted in FIG. 2B , and determining that FIPV is present if said amino acid at amino acid position 1049 is not methionine and/or if said amino acid at amino acid position 1051 is not serine.
  • FIPV feline infectious peritonitis virus
  • a sample comprising feline enteric coronavirus, feline infectious peritonitis virus, feline coronavirus (spike) protein or feline coronavirus nucleic acid can be obtained from any feline directly or indirectly.
  • a sample can for instance be obtained from any feline tissue or fluid or excretion product.
  • Feline tissues, fluids or excretion products from which such sample is obtained include but are not limited to FIP lesions, blood, white blood cells, blood plasma, blood serum, saliva, ascites, urine, faeces, skin, muscle, lymph nodes and liver.
  • a sample according to the invention that is obtained indirectly from a feline may comprise any material that contains feline tissue, fluid or excretion product, such as for instance soil or cat litter.
  • a sample is obtained from a FIP lesion, faeces, blood and/or ascites.
  • a sample is obtained from white blood cells. Blood samples are relatively easy obtained from an animal, and white blood cells are easily isolated from a blood sample subsequently. The present inventors found that in 29 out 31 white blood cell samples obtained from cats in which an alteration of the amino acid at a position corresponding to amino acid position 1049 as depicted in FIG. 2B was detected in a FIP lesion sample, the alteration of said amino acid was also present in the white blood cell sample. Thus, the detection of an alteration of an amino acid is accurately detected in feline white blood cell samples.
  • a feline coronavirus nucleic acid encoding a spike protein whereby the nucleic acid comprises the nucleotide positions 3145, 3146 and/or 3147 and/or the nucleotide positions 3151, 3152 and/or 3153 as depicted in FIG. 2A can be detected in a sample from said feline.
  • a sample from said feline may comprise feline coronavirus nucleic acid, or isolated feline coronavirus nucleic acid.
  • a feline coronavirus nucleic acid comprising nucleotide positions 3145, 3146 and/or 3147 and/or the nucleotide positions 3151, 3152 and/or 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A is amplified before detection.
  • a sample according to the invention may further comprise feline coronavirus or feline coronavirus proteins, including but not limited to the spike protein.
  • the presence of methionine at a position corresponding to amino acid position 1049 of a feline coronavirus as depicted in FIG. 2B is indicative of FECV and the presence of any amino acid other than methionine at said position is indicative of FIPV.
  • alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and/or valine at a position corresponding to amino acid position 1049 of a feline coronavirus as depicted in FIG. 2B is indicative of FIPV.
  • said amino acid other than methionine is leucine.
  • nucleobase adenine at a position corresponding to nucleotide position 3145 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A
  • nucleobase thymine at a position corresponding to nucleotide position 3146 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A
  • nucleobase guanine at the position corresponding to nucleotide position 3147 of the gene encoding a feline coronavirus spike protein as depicted in FIG.
  • nucleobase thymine (t), and/or cytosine (c), and/or guanine (g) at a position corresponding to nucleotide position 3145 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A
  • nucleobases adenine (a), and/or cytosine (c), and/or guanine (g) at a position corresponding to nucleotide position 3146 of the gene encoding a feline coronavirus spike protein as depicted in FIG.
  • nucleobases adenine (a), and/or thymine (t), and/or cytosine (c) at a position corresponding to nucleotide position 3147 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A is indicative of FIPV.
  • Feline coronavirus is an RNA virus. Therefore, when a nucleotide is identified herein as thymine, a uracil is also encompassed by said term, as is known by a skilled person.
  • the invention provides a method according to the invention, wherein the identity of the amino acid at position 1049 is determined by determining a nucleic acid sequence of a feline coronavirus nucleic acid encoding a spike protein, said nucleic acid comprising a nucleotide at, or corresponding to, position 3145, 3146 and/or 3147 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • a cytosine or thymine or guanine at a position corresponding to nucleotide position 3145 of the gene encoding a feline coronavirus spike protein as depicted in FIG.
  • the invention also provides a method according to the invention, wherein the identity of the amino acid at position 1051 is determined by determining a nucleic acid sequence of a feline coronavirus nucleic acid encoding a spike protein, said nucleic acid comprising a nucleotide at, or corresponding to, position 3151, 3152 and/or 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • RNA viruses are RNA viruses.
  • Viral RNA can be isolated and processed with methods known in the art.
  • RNA samples can be freshly prepared from cells or tissues at the moment of harvesting, or they can be prepared from samples that are stored at ⁇ 70° C. until processed for sample preparation.
  • tissues or cell samples can be stored under conditions that preserve the quality of the RNA. Examples of these preservative conditions are fixation using e.g.
  • RNA can for instance be isolated according to the method of Chomczynski and Sacchi (1987) or the method of Boom et al. (1990), or commercially available systems (such as RNeasy total RNA isolation kit from QIAGEN, Germany or High-Pure-RNA-Isolation-Kit® from Roche Diagnostics, Basel, Switzerland).
  • RNA is reverse transcribed into cDNA.
  • Reverse transcriptase polymerase chain reaction (RT-PCR) is for instance performed using specific primers that hybridize to an RNA sequence of interest and a reverse transcriptase enzyme.
  • RT-PCR can be performed with random primers, such as for instance random hexamers or decamers which hybridize randomly along the RNA, or oligo d(T) which hybridizes to the poly(A) tail of mRNA, and reverse transcriptase enzyme.
  • Amplification of nucleotides derived from feline coronavirus, either directly or after RT-PCR can be performed using any nucleic acid amplification method, such as the Polymerase Chain Reaction (PCR; Mullis and Faloona, 1987) or by using amplification reactions such as Ligase Chain Reaction (LCR; Barany, 1991), Self-Sustained Sequence Replication (3SR; Guatelli et al., 1990), Strand Displacement Amplification (SDA; Walker et al., 1992), Transcriptional Amplification System (TAS; Kwoh et al, 1989), Q-Beta Replicase (Lizardi et al., 1988), Rolling Circle Amplification (RCA; U.S. Pat. No.
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reaction
  • SDA Strand Displacement Amplification
  • TAS Transcriptional Amplification System
  • Kwoh et al, 1989 Q-Beta Replicase
  • Rolling Circle Amplification
  • nucleic acid or “nucleic acid molecule” comprises a chain of nucleotides, preferably DNA and/or RNA.
  • primer refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature.
  • An amplification primer is preferably single stranded for maximum efficiency in amplification.
  • a primer is an oligodeoxyribonucleotide.
  • a primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization.
  • a primer pair consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.
  • probe refers to a single-stranded oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative.
  • a specific amplicon detection probe may comprise a label moiety such as a fluorophore, a chromophore, an enzyme or a radio-label, so as to facilitate monitoring of binding of the probes to the reaction product of the amplification reaction.
  • Such labels are well known to those skilled in the art and include, for example, fluorescein isothiocyanate (FITC), [beta]-galactosidase, horseradish peroxidase, streptavidin, biotin, digoxigenin, ⁇ 35>S, ⁇ 14>C, ⁇ 32>P and ⁇ 125>I.
  • FITC fluorescein isothiocyanate
  • [beta]-galactosidase horseradish peroxidase
  • streptavidin biotin
  • biotin digoxigenin
  • a primer or probe according to the invention comprises a nucleic acid sequence, preferably DNA and/or RNA.
  • Said nucleic acid sequence also encompasses other kinds of nucleic acid structures such as for instance a DNA/RNA helix, peptide nucleic acid (PNA), and/or locked nucleic acid (LNA).
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • nucleic acid sequence also encompasses a chain comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks which exhibit the same function as natural nucleotides.
  • Hybridization is known in the art and refers to the combining of complementary, single-stranded nucleic acids, preferably under stringent conditions.
  • complementary or “sequence complementarity” is also known in the art and refers to two nucleic acid strands that can be non-covalently connected by base-pairing.
  • complementary or “substantially complementary” means that two nucleic acid sequences have at least about 70%, preferably about 80%, more preferably 90%, and most preferably about 95%, sequence complementarity to each other. This means that primers and probes must exhibit sufficient complementarity to their template and target nucleic acid, respectively, to hybridise under stringent conditions.
  • primer and probe sequences need not reflect the exact complementary sequence of the binding region on the template and degenerate primers can be used.
  • a non-complementary nucleotide fragment may be attached to the 5′-end of the primer, with the remainder of the primer sequence being complementary to the strand.
  • stringent conditions refers to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamid concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence.
  • the term as used includes reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than to other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances.
  • % sequence identity is defined herein as the percentage of residues in a candidate amino acid sequence or candidate nucleic acid sequence that is identical to the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity.
  • Methods and computer programs for the alignment are well known in the art.
  • One computer program which may be used or adapted for purposes of determining whether a candidate sequence falls within this definition is “Align 2”, authored by Genentech, Inc., which was filed with user documentation in the United States Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991, or the UWGCG Package which provides the BESTFIT program (Devereux et al., 1984).
  • a feline coronavirus nucleic acid comprising a nucleotide corresponding to nucleotide position 3145, 3146 or 3147 and/or the nucleotide positions 3151, 3152 or 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A may be amplified using primers that are capable of hybridizing to at least part of said feline coronavirus nucleic acid sequence.
  • Said primers for instance hybridize to the feline coronavirus nucleic acid sequence encoding a spike protein between a position corresponding to nucleotide position 3055 and a position corresponding to nucleotide position 3669 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • Said primers preferably have a length of between 9 and 50 nucleotides, for instance 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 nucleotides.
  • the nucleic acid product obtained with an amplification method using such primers preferably comprises at least 35 nucleotides, more preferably at least 40 nucleotides, even more preferably at least 50 nucleotides.
  • the invention provides a method according to the invention, further comprising amplifying at least part of a feline coronavirus nucleic acid molecule comprising a region including, or corresponding to, nucleotide position 3145, 3146 and 3147 and/or the nucleotide position 3151, 3152 and 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A using at least one primer which is capable of hybridizing to at least part of said nucleic acid sequence between a position corresponding to nucleotide position 3055 and a position corresponding to nucleotide position 3669 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • a preferred primer pair for use in a method according to the invention comprises a primer which has at least 70% sequence identity with the nucleic acid sequence 5′-CCCTCGAGTCCCGCAGAAACCATACCTA-3′ (SEQ ID NO:1), preferably at least 80% sequence identity with said nucleic acid sequence, more preferably at least 90% sequence identity with said nucleic acid sequence, most preferably at least 95% sequence identity with said nucleic acid sequence, and a primer which has at least 70% sequence identity with the nucleic acid sequence 5′-CAATATTACAATGGCATAATGG-3′ (SEQ ID NO:2), preferably at least 80% sequence identity with said nucleic acid sequence, more preferably at least 90% sequence identity with said nucleic acid sequence, most preferably at least 95% sequence identity with said nucleic acid sequence.
  • Another preferred primer pair for use in a method according to the invention comprises a primer which has at least 70% sequence identity with the nucleic acid sequence 5′-GGCATAATGGTTTTACCTGGTG-3′ (SEQ ID NO:3), preferably at least 80% sequence identity with said nucleic acid sequence, more preferably at least 90% sequence identity with said nucleic acid sequence, most preferably at least 95% sequence identity with said nucleic acid sequence, and a primer which has at least 70% sequence identity with the nucleic acid sequence 5′-TAATTAAGCCTCGCCTGCACTT-3′ (SEQ ID NO:4), preferably at least 80% sequence identity with said nucleic acid sequence, more preferably at least 90% sequence identity with said nucleic acid sequence, most preferably at least 95% sequence identity with said nucleic acid sequence.
  • a primer pair according to the invention comprises a combination of a nucleic acid sequence 5′-CCCTCGAGTCCCGCAGAAACCATACCTA-3′ (SEQ ID NO:1) and a nucleic acid sequence 5′-CAATATTACAATGGCATAATGG-3′ (SEQ ID NO:2), or a combination of a nucleic acid sequence 5′-GGCATAATGGTTTTACCTGGTG-3′ (SEQ ID NO:3) and a nucleic acid sequence 5′-TAATTAAGCCTCGCCTGCACTT-3′ (SEQ ID NO:4).
  • the primer pairs indicated above are used in a nested PCR reaction.
  • a nested polymerase chain reaction two primer pairs are used in successive PCR reactions.
  • the second primer pair is used to amplify a nucleic acid product or part thereof obtained in the amplification reaction using the first primer pair. Therefore, in one embodiment at least part of an amplified nucleic acid, amplified using a first primer pair, is further amplified using a second primer pair.
  • a first primer pair according to the invention comprises, for example, a primer which has at least 70% sequence identity with the nucleic acid sequence 5′-CCCTCGAGTCCCGCAGAAACCATACCTA-3′ (SEQ ID NO:1) and a primer which has at least 70% sequence identity with the nucleic acid sequence 5′-CAATATTACAATGGCATAATGG-3′ (SEQ ID NO:2)
  • a second primer pair according to the invention comprises, for example, a primer which has at least 70% sequence identity with the nucleic acid sequence 5′-GGCATAATGGTTTTACCTGGTG-3′ (SEQ ID NO:3) and a primer which has at least 70% sequence identity with the nucleic acid sequence 5′-TAATTAAGCCTCGCCTGCACTT-3′ (SEQ ID NO:4).
  • a primer pair preferably for identifying feline enteric coronavirus (FECV) or feline infectious peritonitis virus (FIPV), and/or for determining the presence of FIPV or feline infectious peritonitis (FIP) in an animal suspected of suffering from a feline coronavirus infection.
  • FECV feline enteric coronavirus
  • FIPV feline infectious peritonitis virus
  • nucleotide at position 3145, 3146 and/or 3147 and/or the nucleotide at position 3151, 3152 and/or 3153 of the gene encoding a feline coronavirus spike protein can be determined by any method known in the art.
  • ASO allele specific oligonucleotides
  • sequencing of a nucleic acid sequence for example tag-array minisequencing [Fan et al., 2000] or pyrosequencing [Fakhrai-Rad et al., 2002]
  • allele-specific PCR with a blocking reagent ASB-PCR, Morlan et al., 2009
  • OVA oligonucleotide ligation assay
  • OOA oligonucleotide ligation assay
  • MS mass spectrometry
  • qPCR quantitative polymerase chain reaction
  • electronic hybridization for instance matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, Crain and McCloskey 1998)
  • quantitative polymerase chain reaction qPCR
  • electronic hybridization fluorescent single-stranded conformation polymorphism (F-SSCP) analysis
  • F-SSCP fluorescent single-stranded conformation polymorphism
  • DPLC denatur
  • Allele Specific Oligonucleotides are fluorophore-, chromophore-, enzyme- or radio-labelled nucleotide probes which are short and specific for particular RNA or DNA sequences.
  • ASO for instance comprise a nucleotide mutation and only hybridize with nucleic acid sequences comprising this mutation.
  • 2A is for instance detected using a probe that is capable of specifically hybridizing to at least part of said feline coronavirus nucleic acid sequence comprising a nucleotide corresponding to nucleotide position 3145, 3146, and/or 3147 and/or nucleotide position 3151, 3152 and/or 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • Said probe preferably has a length of between 14 and 100 nucleotides, preferably 14, 15, 16, 17, 18, 19, 20, 21, 22, or more nucleotides.
  • a feline coronavirus nucleic acid sequence comprising a nucleotide at, or corresponding to, position 3145, 3146 and 3147 and/or nucleotide position 3151, 3152 and 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A is detected using a probe with a length of at least 14 nucleotides that is capable of specifically hybridizing to at least part of said nucleic acid.
  • a probe is capable of specifically hybridizing to a feline coronavirus nucleic acid comprising cytosine or thymine at a position corresponding to nucleotide position 3145 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • a probe used in a method according to the invention is complementary to a feline coronavirus nucleic acid sequence encoding a spike protein comprising a nucleotide corresponding to nucleotide position 3145, 3146, and 3147 and/or nucleotide position 3151, 3152 and 3153 as depicted in FIG. 2A .
  • coronaviruses are RNA viruses they have relatively high rates of mutation as a skilled person will know. Therefore, the sequence of feline coronaviruses may differ in some nucleotides surrounding nucleotide position 3145, 3146, and 3147 and/or nucleotide position 3151, 3152 and 3153 of the gene encoding a feline coronavirus spike protein.
  • a person skilled in the art knows how a probe according to the invention is modified, for instance by nucleic acid substitution, to enable said probe to hybridize to the nucleic acid sequence of a specific feline coronavirus and detect a nucleotide at, or corresponding to, position 3145, 3146, or 3147 and/or position 3151, 3152 or 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • a preferred probe comprising a nucleotide corresponding to nucleotide positions 3145, 3146, and 3147 as depicted in FIG. 2A for use in a method according to the invention comprises a sequence which has at least 70% sequence identity with the nucleic acid sequence 5′-CCCARRGCCATAGG-3′ (SEQ ID NO:5), wherein R is A or G, preferably at least 80% sequence identity with said nucleic acid sequence, more preferably at least 90% sequence identity with said nucleic acid sequence, most preferably at least 95% sequence identity with said nucleic acid sequence.
  • the invention provides a method according to the invention, wherein said probe comprises the sequence CCCARRGCCATAGG (SEQ ID NO:5).
  • Also provided by the invention is a use of a probe according to the invention, preferably for identifying feline enteric coronavirus (FECV) and/or feline infectious peritonitis virus (FIPV), and/or for determining the presence of feline infectious peritonitis (FIP) in an animal suspected of suffering from a feline coronavirus infection.
  • FECV feline enteric coronavirus
  • FIPV feline infectious peritonitis virus
  • Feline coronavirus nucleic sequences may be determined by sequencing methods known to the skilled person, preferably directly after amplification of relevant nucleic acid. These methods comprise for instance direct double-stranded nucleotide sequencing using fluorescently labeled dideoxynucleotide terminators (Smith et al., 1986), tag-array minisequencing or pyrosequencing. In general such sequencing methods include the isolation of the viral genome nucleic acids by nucleic acid isolation procedures, and the determination of the nucleotide sequence of the isolated nucleic acid, for instance by dideoxy chain termination methods (Sanger et al., 1977) optionally preceded by reverse transcription of RNA into DNA, and/or amplification of the target nucleic acid.
  • At least part of a feline coronavirus nucleic acid sequence comprising a nucleotide corresponding to nucleotide position 3145, 3146 and/or 3147 and/or nucleotide position 3151, 3152 and 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A is sequenced.
  • Oligonucleotide ligation assay is a method for the detection of known single nucleotide polymorphisms. The method is based on the ligation of two adjacent oligonucleotide probes using a DNA ligase while they are annealed to a complementary DNA target.
  • One of the probes is for instance fluorescently labeled and allele-specific.
  • the second probe is a common probe that is complementary to the target DNA sequence immediately downstream (3′) of the site containing the polymorphism, and thus complementary to both alleles.
  • This probe does not need to be fluorescently labeled. Allele discrimination occurs by the ability of DNA ligase to join perfectly matched probes; a 3′ mismatch in the capture probe will prevent ligation.
  • an oligonucleotide ligation assay is used wherein one of the probes is specifically able to hybridize to a feline coronavirus nucleic acid sequence encoding a spike protein comprising an adenine at nucleotide position 3145 as depicted in FIG. 2A which is indicative of FECV.
  • the first nucleotide of a right probe or the last nucleotide of a left probe is a thymine.
  • the second probe is a common probe, which is able to hybridize to both FECV and FIPV nucleic acid sequence starting next to position 3145 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A , for example starting at position 3146 if said second probe is a right probe.
  • FECV FECV
  • FIPV FIPV
  • a feline coronavirus nucleic acid sequence encoding a spike protein, comprising nucleotide position 3145, 3146 and/or 3147 is determined using an oligonucleotide ligation assay (OLA).
  • Real time PCR technology can be used to detect one specific allele of a gene when a blocking reagent is used.
  • This technology is called allele specific PCR with a blocking reagent (ASB-PCR, Morlan et al., 2009).
  • a blocking agent is added to the reaction mixture to prevent amplification of one allele.
  • One of the primers for example the forward primer, is designed as mutant allele specific primer.
  • the other primer is a common primer, which is able to hybridize to both alleles.
  • a blocking agent which is phosphorylated at the 3′ end to prevent amplification, is then designed to bind specifically to the wildtype allele.
  • the blocking agent prevents hybridization of the mutant specific primer to the wildtype allele.
  • a primer set consisting of a common reverse primer and two FIPV nucleic acid specific primers from which the 3′ end nucleotide is complementary to nucleotide position 3145 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A .
  • One of these FIPV specific primers has an adenine at its 3′ end and the other primer has a guanine at its 3′ end which enables said primers to hybridize to a feline coronavirus nucleic acid sequence encoding a spike protein containing a thymine or a cytosine at nucleotide position 3145 respectively.
  • a blocking agent comprising a thymine at the 3′ end can be used, which is able to hybridize to an adenine at nucleotide position 3145.
  • amplification will occur when FIPV nucleic acid is present whereas amplification will not occur when only FECV nucleic acid is present.
  • a feline coronavirus nucleic acid sequence encoding a spike protein, comprising nucleotide position 3145, 3146 and/or 3147 and/or nucleotide position 3151, 3152 and/or 3153 is determined using allele-specific PCR with a blocking reagent (ASB-PCR).
  • MALDI-TOF MS the detection of low (femtomole) quantities of DNA can be achieved.
  • Nucleic acids ranging from 2 to 2000 nucleotides can be detected by using MALDI-TOF MS.
  • MS can be used to analyze mixtures of different nucleic acid fragments without the use of any label because of the mass differences of the nucleobases. Thus, in most cases, separation of nucleic acid fragments is not necessary before MS measurements.
  • a nucleotide of a feline coronavirus nucleic acid sequence encoding a spike protein at nucleotide position 3145 is an adenine, which is indicative of FECV, or a cytosine or thymine, which is indicative of FIPV.
  • the mass of at least part of a feline coronavirus nucleic acid sequence, said part comprising a nucleotide corresponding to nucleotide position 3145, 3146, and/or 3147 and/or nucleotide position 3151, 3152 and/or 3153 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A is determined.
  • the mass of said nucleic acid sequence is determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
  • an amino acid in a feline coronavirus amino acid sequence encoding a spike protein at amino acid position 1049 and/or amino acid position 1051 as depicted in FIG. 2B can be detected using any method known in the art. Such amino acid is for instance detected using antibodies or functional equivalents thereof, mass spectrometry or Edman degradation reactions.
  • a coronaviral protein can be purified with methods known in the art. For instance, coronaviral protein can be purified using gel electrophoresis or chromatography methods, such as affinity chromatography.
  • a functional equivalent of an antibody is defined as a compound which has at least one same property as said antibody in kind, not necessarily in amount. Said functional equivalent is capable of binding the same antigen as said antibody, albeit not necessarily to the same extent.
  • a functional equivalent of an antibody preferably comprises a single domain antibody, a single chain antibody, a nanobody, a unibody or a single chain variable fragment (scFv).
  • a functional equivalent of an antibody is for instance produced by altering an antibody such that at least one property—preferably an antigen-binding property—of the resulting compound is essentially the same in kind, not necessarily in amount. This is done in many ways, for instance through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning is likely not to be seriously affected.
  • An immunogenic part of a feline coronavirus comprising a feline coronavirus spike protein is defined as a part which has at least one property in common with a feline coronavirus comprising a feline coronavirus spike protein in kind, though not necessarily in amount.
  • An immunogenic part of a feline coronavirus spike protein is defined as a part which has at least one same property as a feline coronavirus spike protein in kind, not necessarily in amount.
  • Said immunogenic part is preferably capable of eliciting an immune response against a feline coronavirus, preferably a feline infectious peritonitis virus (FIPV), in an animal.
  • FIPV feline infectious peritonitis virus
  • amino acid of a feline coronavirus spike protein amino acid sequence corresponding to amino acid position 1049 and/or amino acid position 1051 as depicted in FIG. 2B is for instance detected using an antibody or functional equivalent that is specifically directed against an epitope of a feline coronavirus spike protein that comprises amino acid position 1049 and/or amino acid position 1051 as depicted in FIG. 2B .
  • Said amino acid position 1049 and/or amino acid position 1051 as depicted in FIG. 2B enables discrimination between FECV and FIPV.
  • a methionine at amino acid position 1049 is indicative of FECV, whereas any amino acid other than methionine at this position, preferably leucine, is indicative of FIPV and any amino acid other than serine at amino acid position 1051, preferably alanine, in indicative of FIPV. Therefore, the invention provides a method according to the invention, wherein an amino acid of a feline coronavirus spike protein at a position corresponding to amino acid position 1049 and/or amino acid position 1051 as depicted in FIG. 2B is detected by using an antibody or functional equivalent thereof specifically directed against an epitope of a FIPV spike protein encompassing amino acid 1049 and/or amino acid 1051.
  • said epitope comprises an amino acid other than methionine at a position corresponding to amino acid position 1049 and/or said epitope comprises an amino acid other than serine at a position corresponding to amino acid position 1051 as depicted in FIG. 2B .
  • An antibody or functional equivalent thereof specifically directed against an epitope of a FIPV spike protein, which epitope comprises an amino acid corresponding to amino acid position 1049 and/or amino acid position 1051 as depicted in FIG. 2B can be detected with any method known in the art.
  • said antibody or functional equivalent thereof is fluorophore-, chromophore- or enzyme-labelled, and can thus be detected with for instance fluorescence microscopy or spectrophotometry.
  • An antibody or functional equivalent can also be detected using a second antibody which is for instance fluorophore-, chromophore- or enzyme-labelled.
  • Such labels are well known to those skilled in the art and include, for example, fluorescein isothiocyanate (FITC), [beta]-galactosidase, horseradish peroxidase, streptavidin, biotin or digoxigenin.
  • FITC fluorescein isothiocyanate
  • [beta]-galactosidase horseradish peroxidase
  • streptavidin horseradish peroxidase
  • biotin or digoxigenin digoxigenin
  • an antibody or functional equivalent specifically directed against an epitope of a FIPV spike protein which epitope comprises an amino acid other than methionine at a position corresponding to amino acid position 1049 as depicted in FIG. 2B and a kit of parts comprising an antibody according to the invention and means for detecting said antibody.
  • MALDI-TOF MS the detection of low quantities of amino acid sequences can be achieved. MS can be used to analyze mixtures of different amino acid sequences without the use of any label because of the mass differences of the amino acid sequences. Thus, in most cases, separation of amino acid sequences is not necessary before MS measurements. Using MALDI-TOF MS it is for instance possible to discriminate between a feline coronavirus amino acid sequence comprising a methionine at amino acid position 1049 as depicted in FIG. 2B , and a feline coronavirus amino acid sequence comprising an amino acid other than methionine at amino acid position 1049 as depicted in FIG. 2B .
  • the mass of at least part of a feline coronavirus amino acid sequence is determined.
  • the mass of said amino acid sequence is determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
  • the invention provides an immunogenic composition
  • a feline coronavirus spike protein or immunogenic part thereof comprising an amino acid other than methionine at a position corresponding to amino acid position 1049, and/or an amino acid other than serine at a position corresponding to amino acid position 1051 as depicted in FIG. 2B , or a spike protein encoding feline coronavirus nucleic acid, comprising a cytosine or thymine at a position corresponding to nucleotide position 3145, and/or a guanine at a position corresponding to nucleotide position 3151 as depicted in FIG.
  • an immunogenic composition according to the invention is used as a vaccine.
  • feline coronavirus spike protein or immunogenic part thereof comprising an amino acid other than methionine at a position corresponding to amino acid position 1049, and/or an amino acid other than serine at a position corresponding to amino acid position 1051 as depicted in FIG. 2B , or a spike protein encoding feline coronavirus nucleic acid comprising a cytosine or thymine at a position corresponding to nucleotide position 3145, and/or a guanine at a position corresponding to nucleotide position 3151 as depicted in FIG.
  • a feline coronavirus comprising a nucleic acid comprising an adenine at a position corresponding to nucleotide position 3145, and/or a thymine at a position corresponding to position 3151 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A , or a feline coronavirus comprising a feline coronavirus spike protein or immunogenic part thereof comprising a methionine at a position corresponding to amino acid position 1049, and/or a serine at a position corresponding to amino acid position 1051 as depicted in FIG. 2B , or any combination thereof, for eliciting an immune response against a feline coronavirus, preferably a feline infectious peritonitis virus (FIPV), in a feline.
  • FIPV feline infectious peritonitis virus
  • One embodiment provides a use of a feline coronavirus spike protein or immunogenic part thereof comprising an amino acid other than methionine at a position corresponding to amino acid position 1049, and/or an amino acid other than serine at a position corresponding to amino acid position 1051 as depicted in FIG. 2B , or a spike protein encoding feline coronavirus nucleic acid comprising a cytosine or thymine at a position corresponding to nucleotide position 3145, and/or a guanine at a position corresponding to nucleotide position 3151 as depicted in FIG.
  • a feline coronavirus comprising a nucleic acid comprising an adenine at a position corresponding to nucleotide position 3145, and/or a thymine at a position corresponding to position 3151 of the gene encoding a feline coronavirus spike protein as depicted in FIG. 2A
  • a feline coronavirus comprising a feline coronavirus spike protein or immunogenic part thereof comprising a methionine at a position corresponding to amino acid position 1049, and/or a serine at a position corresponding to amino acid position 1051 as depicted in FIG.
  • an immunogenic composition or prophylactic agent for eliciting an immune response against a feline coronavirus, preferably a feline infectious peritonitis virus (FIPV), in a feline.
  • a feline coronavirus preferably a feline infectious peritonitis virus (FIPV)
  • FIPV feline infectious peritonitis virus
  • FIG. 1 Schematic representation of the feline coronavirus RNA genome.
  • the 5′ part (left) specifies the precursors encoding the replication and transcription functions derived from open reading frames (ORFs) 1a and 1b. Downstream thereof, towards the 3′ end, the genes for the structural proteins S (spike protein), E (envelope protein), M (membrane protein) and N (nucleocapsid protein), and for the accessory proteins 3a, 3b, 3c, 7a and 7b are located.
  • ORFs open reading frames
  • FIG. 2 A) Nucleotide sequences of the feline coronavirus spike gene (nucleotides 1-4407, SEQ ID NO:6), corresponding to nucleotides 20395-24801 of a feline coronavirus as defined in the nucleotide sequence of NC_012955 (Feline coronavirus UU10, complete genome) and nucleotides 20382-24788 (SEQ ID NO:7) of a feline coronavirus as defined in the nucleotide sequence of NC 012952 (Feline coronavirus UU8, complete genome).
  • FIG. 3 Agarose gel electrophoresis of amplified RNA from 6 clinical samples obtained from faeces of infected cats. Lane M is a molecular size standard, lanes 1-6 are the clinical samples and lane 7 is a negative control.
  • FIG. 4 A) Alignment of partial sequences of faeces or plasma derived FCoV RNA isolated from 42 healthy cats and five partial sequences derived from samples of FIP-confirmed cats, i.e. Q093501030_326B_4546.scf (white blood cell derived), Q093501032_327B_4546.scf (white blood cell derived), Q093501036_321S_4546.scf (serum derived), Q093501038_321A_4546.scf (ascites derived) and Q093501046_K11_019.ab1 (white blood cell derived) (SEQ ID NO:10-104); B) Alignment of partial sequences of lesion-derived FCoV RNA isolated from 54 FIP-confirmed cats (SEQ ID NO:105-210); C) Alignment of partial sequences of faeces-derived FCoV RNA isolated from FIP-confirmed cats
  • Primers were designed using the FCoV genome sequences with accession numbers of NC_012955 and NC_012952. Amplifications was performed using 30 cycles of 94° for 60 s, 50° for 30 s, and 72° for 1 min and additional extension at 72° for 7 min at the end of amplification. The PCR fragments were isolated and purified from agarose gel after electrophoresis using the Qiagen gel Extraction kit (Qiagen Benelux B.V., Venlo, The Netherlands). Sequencing was performed by BaseClear Holding B.V. (Leiden, The Netherlands).
  • a 601-bp fragment was obtained only in one clinical sample, as is seen in lane 2 of FIG. 3 .
  • a 139-bp fragment was amplified when the nested primers were applied on the products of the 1st run RT-PCR. Now a product was seen not only in lane 2, but also with the amplified RNA's shown in lanes 3, 5 and 6.
  • Genomic RNA extraction, cDNA synthesis, amplification and sequencing were performed according to the materials and methods of example 1.
  • FIG. 4A contains five sequences derived from samples of cats with confirmed FIP (Q093501030_326B_4546.scf, Q093501032_327B_4546.scf, Q093501036_321S_4546.scf, Q093501038_321A_4546.scf and Q093501046_K11_019.ab1), meaning that in 42/42 faeces or plasma derived FCoVs from healthy cats a methionine was present at amino acid position 1049.
  • Genomic RNA extraction, cDNA synthesis, amplification and sequencing were performed according to the materials and methods of example 1.
  • faeces samples were FCoV positive.
  • a nucleic acid sequence encoding a methionine at amino acid position 1049 and a serine at amino acid position 1051 was detected in all (137) faeces-derived FCoVs from healthy cats.
  • EDTA-blood samples from 89 healthy or non-FIP suspected cats were obtained and separated into white blood cells (WBC) and plasma. Serum samples from 56 healthy or non-FIP suspected cats were obtained.
  • WBC white blood cells

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